Monocotyledonous plants are more difficult to transform than dicotyledonous plants, in spite of their importance in agriculture. The conventional transformation of these plants has been carried out by various forced introduction methods such as those using gene guns for intact cells, or electroporation or PEG (polyethylene glycol) method for protoplasts.
However, there are problems in the electroporation method or PEG method that regeneration from protoplasts to normal plants is difficult, and in case of gene gun, that expensive facilities are required and a large number of samples can not be processed simultaneously. Furthermore, a large gene fragment of 10 kb or more into monocotyledonous plants has not been introduced without its rearrangement.
Recently, Hiei et al. have reported that rice, a typical monocotyledonous crop can be transformed with a pBI system vector and Agrobacterium (Hiei et al., Plant Journal, 6:271-282 (1994)). This has made the transformation of rice drastically easy.
However, the pBI vector used by Hiei et al. can stably introduce only about 10 kb DNA into rice. If larger DNA was integrated into such a shuttle vector, the vector could not be stably maintained even in E. coli.
By the recent advances of genome studies, the positional cloning method to isolate a gene, which is based on its positional information on the chromosome map, became the focus of interest as a promising method to isolate the useful gene determining agricultural traits. In order to apply this technique to identify genes causing these traits from the candidate genes selected by using their map information, the trait expression by the gene in the genome fragment must be confirmed by transformation of the fragment to plant. In this case, introduction of the larger genome fragment makes the screening of genes the more efficient. Therefore, it is desirable to introduce as large a genome fragment as possible.
Although the binary cosmid method and PEG method are known to introduce genome fragments of 10 kb or larger size into a plant, both of the methods have deficiencies as follows:
(1) As for the binary cosmid method, the upper size of a genome fragment to be introduced is limited up to about 20 kb, and pBI system RK2 is used as replication origin. Therefore, it has been often difficult to stably maintain the plasmid even in E. coli into which the plasmid with the chromosome fragment is to be introduced at first. PA1 (2) As for the PEG method, the regeneration from protoplasts requires high proficiency of the skill, and often the introduced fragments are severely rearranged making normal complementation by the fragment rather difficult. Therefore, such methods are not sufficiently suited to assay the functions of large genome fragments.